Biological imager

ABSTRACT

Apparatus and method for quantifying the biological components of a biological system having at least two different refractive indices through detection of wave front distortions. The biological component fractions are determined based on information gathered on their respective indices when exposed to particular wavelengths of light.

BACKGROUND OF THE INVENTION

[0001] 1. Filed of the Invention

[0002] This invention relates to an apparatus and method for analyzingdifferent components in a system having at least two differentrefractive indices. This invention further, and more particularly,relates to biological imaging through the components of a biologicalsystem.

[0003] 2. Background Art

[0004] In many situations the monitoring of a biological system in realtime is desired in addition to determining the biological componentswithout using a computer-intensive technique.

[0005] It is desirable to make measurements without having torecalibrate each time a measurement is taken, such as those that useabsorption techniques. Different material properties requirerecalibration of currently used equipment. This invention addresses thisproblem.

BRIEF SUMMARY OF THE INVENTION

[0006] This invention relates to an apparatus and method for analyzing asystem using the refractive index of light. The biological componentfractions of a biological system are determined using the refractiveindex of materials in relation to specific wavelengths of light.

[0007] The invention can determine the percentages of biologicalcomponents and water without prior knowledge of the refractive index ofthe biological components. The method determines the percentages ofbiological component fractions, include passing a focused light beamthrough the biological components, measuring the displacement of thepoint of focus from a known focal point with a known index ofrefraction, and thereby calculating the percentages of biologicalcomponents present.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0008]FIG. 1a is a diagram of the biological imager of this invention.

[0009]FIG. 1b is a diagram of another biological imager.

[0010]FIGS. 2 and 2a show schematic diagrams of the biological imager.

[0011]FIGS. 3a-3 c show schematic diagrams of the biological imager.

[0012]FIG. 4 is a schematic diagram of one embodiment of the biologicalimager with an area sensor and lens array.

[0013]FIG. 5a is a schematic of the biological imager with a referencefluid.

[0014]FIG. 5b is a schematic of the biological imager with the referencematerial and another lighter fluid.

[0015]FIG. 5c is a schematic drawing of the biological imager with withthe reference material and another heavier fluid.

[0016]FIG. 5d is a schematic of the biological imager with the referencematerial and with both a lighter and a heavier fluid.

[0017]FIG. 5e is a schematic drawing of the biological imager and aShack-Hartmann detector.

[0018]FIG. 6 is an embodiment with an imaging lens.

[0019]FIG. 7 shows a schematic drawing of the strobe and sensorarrangement.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Virtually every biological system consists of a variety ofcomponents composed of fluids and gels that exist as a mixture, eachcomponent with one or more distinct refractive indices when a specificwavelength of light passes through the mixture. Typically elaborateimaging methods must be used to image these biological systems and tomeasure their physical properties such as viscosity, geometry, relativefractions, and flow rates if needed.

[0021] Fluids and gels, specifically those of different biologicalcomponents, refract light by varying degrees when a specific wavelengthpasses through the mixture. The amount of refraction is a function offluid composition and wavelength of the light passing through the fluid.The refractive index is a physical property of the fluid and is aparameter for determining the optical interaction of the fluid and thelight refracted through it.

[0022] This invention is applicable to all systems including biologicalsystems. For purposes of brevity, however, the description herein willbe primarily directed to invitro biological systems, particularly a cellwith components composed of protein matrix-based gels.

[0023]FIG. 1a shows a biological imager 10 deployed to analyze abiological mixture of biological components including water, particulatematter, and other materials that could be found in a biological system.FIG. 1 shows a cell 11 with a variety of components such as protein,matrix-based gels hereafter referred to as a biological mixture 12 whichcould be any system of components but is shown here as a cell. Controlof the fluid of the bath, as well as its movement if relevant, is knownand can be described in a variety of ways, some of which are not to bediscussed in this application.

[0024] The biological imager 10 has a light source 14 and a detector 16arranged on opposite sides of a sample of a biological mixture 12 whichis made up of non-immiscible biological components. This mixture must besuch that when separated it retains its ratio. The biological imager issuch that there are transparent, or partially transparent, openings 18between the light source and the detector that allow light to pass fromthe light source through the biological mixture 12 to detector 16. Thebiological imager 10 can incorporate any number of optical elements,including but certainly not limited to lenses, filters, diffractiongratings, and other optical elements that will be discussed in detaillater. These optical elements can be incorporated into the openings 18or can stand alone.

[0025] The light source 14 is a point source or extended point sourcewith one or more discrete wavelengths temporally and/or spatiallyseparated such as would be true for a single source that is pulsed orone or more spatially separated sources. The source can include one ormore discrete wavelengths or be a filtered white light source. If thereare two or more light sources they can have overlapping spectra but thewavelengths must be at least detectable so that there is sufficientenergy that is unique to each wavelength to provide two uniquerefractive properties after the light has passed through the fluidmixture. Note that alternatively a wideband white light source could beused unfiltered (without discrete wavelengths detectable at the source)and filtered at the detector. What is required is that the twowavelengths must be discrete to provide distinct and separateinformation when separately focused. Each discrete wavelength will beseparately focused and the shift in the focal point measured from aknown focal point.

[0026]FIG. 1b shows a biological imager 10 a to analyze the fluidmixture 12 where the detector 16 is in an alternate location. Thebiological imager 10 a has a second surface 19 that can incorporate thedetector 16 or may be reflective or partially reflective such that thedetection of a component may be directly read, recorded on the surface19 or reflected toward another location. This embodiment couldincorporate a circuit that diverted the focal point electronically ascould the other embodiments.

[0027]FIGS. 2 and 2a are detailed schematic diagrams of the biologicalimager 10 shown in a container 20 which could be a laboratory. The fluidmixture 12 is shown between the light source 14 and the detector 16. Inthis embodiment there is a first quadradric phase plate (L₁) 22 and asecond quadradric phase plate (L₂) 24 both of which preferably arepositive lenses, and hereafter referred to as first lens 22 and secondlens 24. Light from the source 14 can be focused in the fluid mixture 12where a real image (I₁) of the source 14 is formed by L₁. The lighttravels on to (L₂) which can form another image (I₂) near an aperture orspatial filter 26 before being focused by a third collimating lens 28onto the lens array 30 and an area sensor 32 which could be a focalplane array. It is not necessary that the focus occur in the fluidmixture 12. The volume of the fluid mixture 12 that is being analyzedwill be referred as the analysis zone 34 in the following discussion.The analysis zone is also referred to as a capturing cone. The fact thatthis covers a larger volume allows integration and averaging of a largervolume of fluid mixture 12.

[0028]FIG. 3a; FIG. 3b, and FIG. 3c, show alternate arrangements of alight source 14 and the detector 16 as well as one or more lenses thatwould work under certain circumstances. FIG. 3a has the first lens 22,the aperture 26, and the collimating lens 28. FIG. 3b does not have thecollimating lens 28 and so the detector 16 must be able to handle lightthat has not been collimated. In this scenario, it may be more difficultto determine a unique solution due to the presence of higher orderdistortions. The same would be true if the collimating lens 28 waspresent but the aperture 26 was removed. The aperture 26 is not requiredin certain circumstances. FIG. 3c adds a filter 35 so that a white lightsource can be used without a filter at the source but with some sort offilter at the detector 16. The detector filter could even be anelectronic device or involve an algorithm.

[0029]FIG. 4 shows the light source 14 directed toward the firstcollimating lens (L₁) 22 which in this case is shown to be at a distancethat is twice its focal length from the light source. The first lenscould be any distance from the light source as would be known in the artas long as the expanding wave front is known as it enters the biologicalmixture 12. The wave front will be refracted by the first lens 22,refracted through the biological mixture 12, and in this embodiment, ifrefracted through pure water, would focus at a point 36 between thefirst lens 22 and the second lens 24. The focus point 36, if it was purewater, would be N_(water) (refractive index of water)·2·f₁ (focal lengthof the first lens 22) from the first lens 22, and a distance equal toN_(water) (refractive index of water)·2·f₂ (focal length of the secondlens 24) from the second lens 24. The lenses 22 and 24 are separated bya distance “d” shown by 38. The emerging light would be refracted by thesecond lens 24 and directed toward the spatial filter 26, which in thisembodiment is a distance equal to 2·f₂ from the second lens 24. Afterpassing through the biological imager 10 the light wave front has beendistorted by scattering in the fluid. The distorted wave frontrepresented by 40 in the diagram would defocus by higher order termsincorporated in it, as shown in the diagram by the wavy line 42. Afterthis distorted wave front 40 passes through the spatial filter oraperture 26, the wave front has some of the noise eliminated leavingbiological fluid mixture dependent defocus. The choice of an aperture orspatial filter 26 is critical to the success of this apparatus because,like a confocal microscope, it eliminates noise (higher orderdistortions) without removing the focus information. If the aperture istoo small, the information that includes the mixture dependent focuswould be lost; but if the aperture is too large, unnecessary noise woulddetract from the efficiency of the apparatus. All of the distances mustbe measured precisely since the shift in the focal point will be theorder of a wavelength.

[0030] The filter aperture requirements (size, geometry, etc.) areheavily dependent on the optical system layout and the definedmeasurement tolerances. Given that defocus shifts are the primarywavefront aberration to be measured, all other contributions to the WFE(wavefront error) can be ignored. The filter aperture 26 can help reducethe other aberrations (typically, of a higher order than defocus), whichare primarily due to scattering generated by the material beingmeasured. A basic review of how to deal with such things can be found inGoodman's book “Introduction to Fourier Optics”, in chapter and section:“Frequency analysis of optical imaging systems, Aberrations and theireffects on frequency response” (Chapter 6-4 in the 1^(st) edition).Here, the generalized exit pupil function is defined as:

P(x_(p),y_(p))=p(x_(p),y_(p))exp(jkW(x_(p),y_(p))), where p(x_(p),y_(p))

[0031] is the non-aberrated pupil function applied to the image ataperture 26. W(x_(p),y_(p)) encompasses the aberration phase terms ofthe exit pupil wavefront. Assuming defocus is the dominant term we have:${{W\left( {x_{p},y_{p}} \right)} = {\frac{ɛ\left( {x_{p}^{2} + y_{p}^{2}} \right)}{2} + {{Higher}\quad {order}\quad {terms}}}},$

[0032] where ε is the phase error term. The specified shifts in defocusare related to ε and an aperture 26 can be constructed such that thehigher order contributions are minimized with respect to the desiredmeasurable defocus range.

[0033] In this embodiment the third collimating lens 28 (also referredto as “a fourier transform lens” or “FT lens”) is placed a distanceequal to its focal length from the spatial filter 26. The third,collimating lens 28 essentially turns the wave front “inside out” andthe focus information is the largest component of the light wavefrontleaving the collimating lens 28. The light is focused on the lens array30 of this embodiment which could take many different formats (such asShack-Hartmann, Interferometry phase diversity, various algorithms,electric circuits, etc.). A Shack-Hartmann area sensor 32 can performinverse fourier transform resulting in spot shifts when a refractiveindex of the biological mixture 12 changes. If the parameters arecarefully chosen and tuned so that there is no shift when the medium iswater, and there is a positive shift when there is the presence ofcertain components and there is a negative shift when there is thepresence of other components allowing a simple deflection measurement todetermine the fraction of certain components in a sample. The areasensor 32 could take another format such as interferometer, which wouldrequire the transmission of an undistorted wavefront from the lightsource 14 to the detector 16 to the area sensor 32 in order to get theinterference necessary for the interferometer to work. In which case,there would be no need for the collimating lens 28.

[0034]FIG. 5a is a schematic diagram of the biological imager 10 and areference fluid with a known refractive index such as water, calibratedso that the focus of the light passed through at the detector 16.

[0035]FIG. 5b is a schematic drawing of the biological imager 10 andboth the reference fluid and another lighter fluid such that the focalpoint changes in relation to the change in refractive index due to theamount of biological components in the mixture.

[0036]FIG. 5c is a schematic drawing of the biological imager 10 andboth the reference fluid and another heavier fluid such that the focalpoint changes in relation to the change in refraction index due to theheavier fluid. Note that the focal point will shift in a directionopposite of that in FIG. 4b in this example. The introduction of thelighter gas causes less refraction because the light is travelingthrough a fluid with a lower refractive index.

[0037]FIG. 5d is a schematic drawing of the biological imager 10 and thereference fluid, as well as both a lighter and a heavier fluid so thatthere is the need to focus two different wavelengths of light to solvefor the two unknown fractions of biological components present.

[0038]FIG. 5e is a schematic drawing of the biological imager 10 withall three phases of fluid and a Shack-Hartmann detector.

[0039] The biological imager 10 of FIG. 6 is set to analyze a fluid flowof a biological mixture. This biological system could be part of anorganism. A sample from an organism, or it could be in a separate vesselor system in a laboratory. This is particularly effective in organismsbecause of the gel-like nature of living cytoplasm, the interior livingcells. Many cellular functions can be attributed to and are accomplishedby gel properties of sub-membrane cytoskeleton or actin, microtubulesand other protein structures such as regulating ionic fluxes andconcentrations. Cytoplasmic gels manifest collective phase transitionssuch as ploymerization of actin proteins with accompanying ordering ofcell water and exclusion of large cations. These collective phasetransitions can explain not only ionic fluxes, but also voltagegradients, propagating action potentials, mitosis, muscle contractionand cell movement. The fact that cells include and are regulated bycomponents such as the protein matrix-based gels make this inventionparticularly useful. Since the cytoplasm is intrinsically reactive andable to maintain cell homeostasis and functions, the cytoplasm gel bestcaptures the essence of the living state and can be measured in responseto the refractive index of light in accordance with this invention.

[0040]FIG. 7 shows a fluid stream that can be analyzed using thisinvention to determine the rate of flow.

[0041] Any properties that can be derived from the different refractiveindices of the cellular components, particularly protein matrix-basedgels. Properties such as the geometry of the structure in a bath ofknown refractive index using the refractive index of light is possiblebecause the structure will refract light at the boundaries.

[0042] One embodiment of the method for measuring the biologicalfractions includes projecting two discrete wavelengths λ₁ and λ₂ throughthe biological components causing wavefront distortion allowing for thedetermination of two separate focal point displacements and thedetermination of two biological fractions in response to themeasurements generated by λ₁ and λ₂. This method requires values of λ₁and λ₂ such that:

[0043] (a) λ₁: chosen such that N_(BC) (λ₁)≠N_(water) (λ₁); N_(BC)(λ₁)≠N_(air) (λ₁); and

[0044] (b) λ₂: chosen such that N_(BC) (λ₂)≠N_(water) (λ₂); N_(BC)(λ₂)≠N_(air) (λ₂).

[0045] In order to solve for one unknown, for example the fraction ofDNA, the following equation is solved where:

[0046] (a) OPL=Optical Path Length (measured by the refractometer)

[0047] (b) OPL[measured]=N_(avg) (λ);

[0048] (c) AN_(BC1)(λ₁)+BN_(BC2)(λ₁)+CN_(BC3)(λ₁)=N_(avg)(λ₁);

[0049] (d AN_(BC1)(λ₂)+BN_(BC2)(λ₂)+CN_(BC3)λ₂)=N_(avg)(λ₂);

[0050] (e) A+B+C=1

[0051] (f) N(λ)=refractive index.

[0052] In order for these equations to be solved, it is necessary thatthe fluid components do not chemically interact. This is characterizedby being able to be separated with the component ratios preserved. Inone example, when pure water is a reference point, the focal pointchanges as a function of the material in the flow stream. The light beamwill curve (spread) when compared to the reference. This curvature canbe measured. There are a number of combinations that can be solved forincluding a component refractive index or the ratio of components. Ifthere are two or more unknowns then additional wavelengths like λ₁ andλ₂ will be required to solve for these additional unknowns.

[0053] First the first λ₁ is focused and the distance from the knownfocus in water measured so that N_(avg) (λ₁) can be calculated.Subsequently, the second λ₂ is focused, the distance from the knownfocus measured, and N_(avg) (λ₂) calculated. With all but A, B, and Cknown the coefficients A, B, and C can be calculated from the threeequations.

[0054] If the refractive index of one biological component (N_(BC2)) isalso unknown but the refractive indices of two other biologicalcomponents (N_(BC1) and N_(BC3)), are known, then there are fourunknowns (A, B, C and N_(BC2)) since only N_(BC1) and N_(BC3) are known.To solve these equations, four wavelengths (λ₁, λ₂; λ₃, λ₄) must befocused and the distance from a known focal point measured for each[N_(avg) (λ₁); N_(avg) (λ₂), N_(avg) (λ₃), N_(avg) (λ₄)]. The N_(BC2)varies in a known way according to the Cauchy relationship such that:

[0055] N(λ₁)∝K₁+K₂N(λ₁ ²), where the higher order terms are ignored, andthen N_(oil), A, B, and C can be solved for simultaneously. Includingadditional terms in the Cauchy expansion will require additionalwavelengths in order to find a solution.

[0056] A detailed analysis using the physical arrangement shown in FIG.2 follows:

[0057] a) Dimension Items

[0058] (i) s_(o1)—Distance of light source (14) to the 1^(st) principleplane of Lens 1 (22)

[0059] (ii) s_(i1)(N_(ave)(λ))—Distance of imaged light source (I₁) tothe 2^(nd) principle plane of Lens 1 (22) for N_(ave) (λ)

[0060] (iii) s_(o2)(N_(ave)(λ))—Distance of imaged light source (I₁) to1^(st) principle plane of Lens 2 (24) for N_(ave)(λ)

[0061] (iv) s_(i1ref)(N_(ref)(λ_(ref)))—Reference distance of imagedlight source (I₁) to 2^(nd) principle plane of Lens 1 (22) forN_(ref)(λ_(ref))

[0062] (v) s_(o2ref)(N_(ref)(λ_(ref)))—Reference distance of imagedlight source (I₁) to 1^(st) principle plane of Lens 2 (24) forN_(ref)(λ_(ref))

[0063] (vi) Δs_(i1)(λ)—Change in s_(i1) relative to reference ats_(i2ref) due to wavelength and material changes between Lens 1 (22) andLens 2 (24)

[0064] (vii) d—Thickness of the material to be analyzed

[0065] (viii) s_(i2)(N_(ave)(λ))—Distance of imaged light source (12) tothe 2^(nd) principle plane of Lens 2 (24) for N_(ave)(λ)

[0066] (ix) s_(i2ref)(N_(ref)(λ_(ref)))—Reference distance of imagedlight source (I₂) to 2^(nd) principle plane of Lens 2 (24) forN_(ref)(λ_(ref))

[0067] (x) Δs_(i2)(N_(ave)(λ))—Change in s_(i2) relative to reference ats_(i2ref) due to wavelength and material changes between Lens 1 (22) andLens 2 (24)

[0068] (xi) f₃—Effective focal length of Lens 3 (28)

[0069] (xii) α—Aperture size

[0070] (xiii) s_(o3)(N_(ave)(λ))13 Distance of imaged light source (12)to 1^(st) principle plane of Lens 3 (28) for N_(ave)(λ)

[0071] (xiv) z—Distance from aperture 26 to where the WFE (wavefronterror) is measured

[0072] (xv) y—Distance perpendicular from optical center line to wherethe WFE is measured

[0073] (xvi) WFE(λ)—Paraxial Wavefront Error (measured in waves ofλ_(ref)) relative to reference due to wavelength and material changesbetween Lens 1 (22) and Lens 2 (24)

[0074] b) Glossary:

[0075] (i) λ≡Wavelength

[0076] (ii) λ_(ref)≡Reference wavelength

[0077] (iii) N≡Refractive index

[0078] (iV) ƒ≡Effective focal length for all λ to be used in device,where ƒ>0 for all lenses

[0079] (v) N_(ref)(λ_(ref))≡Index of a reference component (m=0) at areference wavelength (λ_(ref))

[0080] (vi) A_(m)≡Solution component volume percentage

[0081] (vii) n≡Number of solution components

[0082] c) Known Terms:

[0083] (i) λ_(ref), N_(ref)(λ_(ref)), ƒ₁, ƒ₂, ƒ₃, s_(o1), and d

[0084] d) Equations: $\begin{matrix}(i) & {{N_{ave}(\lambda)} = {{N_{ref}\left( \lambda_{ref} \right)} + {\Delta \quad {N_{ave}(\lambda)}}}} \\\quad & {\quad {= {\sum\limits_{0}^{n - 1}{A_{m}{N_{m}(\lambda)}}}}} \\({ii}) & {{s_{refi2}(\lambda)} = \frac{{df}_{2} - {{N_{ref}\left( \lambda_{ref} \right)}f_{1}f_{2}{s_{o1}/\left( {s_{o1} - f_{1}} \right)}}}{d - {{N_{ref}\left( \lambda_{ref} \right)}\left\lbrack {f_{2} - {f_{1}{s_{o1}/\left( {s_{o1} - f_{1}} \right)}}} \right\rbrack}}} \\({iii}) & {{s_{i2}(\lambda)} = \frac{{df}_{2} - {{N_{ave}(\lambda)}f_{1}f_{2}{s_{o1}/\left( {s_{o1} - f_{1}} \right)}}}{d - {{N_{ave}(\lambda)}\left\lbrack {f_{2} - {f_{1}{s_{o1}/\left( {s_{o1} - f_{1}} \right)}}} \right\rbrack}}} \\({iv}) & {{\Delta \quad {s_{i2}(\lambda)}} = {{s_{refi2}(\lambda)} - {s_{i2}(\lambda)}}} \\\quad & {\quad {= {\frac{{df}_{2} - {{N_{ref}\left( \lambda_{ref} \right)}f_{1}f_{2}{s_{o1}/\left( {s_{o1} - f_{1}} \right)}}}{d - {{N_{ref}\left( \lambda_{ref} \right)}\left\lbrack {f_{2} - {f_{1}{s_{o1}/\left( {s_{o1} - f_{1}} \right)}}} \right\rbrack}} -}}} \\\quad & {\quad \frac{{df}_{2} - {{N_{ave}(\lambda)}f_{1}f_{2}{s_{o1}/\left( {s_{o1} - f_{1}} \right)}}}{d - {{N_{ave}(\lambda)}\left\lbrack {f_{2} - {f_{1}{s_{o1}/\left( {s_{o1} - f_{1}} \right)}}} \right\rbrack}}}\end{matrix}$

[0085] (v) s_(o3)(λ)=ƒ₃−Δs_(i2)(λ) $\begin{matrix}({vi}) & {{s_{i3}(\lambda)} = \frac{s_{o3}f_{3}}{s_{o3} - f_{3}}} \\\quad & {\quad {= {f_{3} - \frac{f_{3}^{2}}{\Delta \quad {s_{i2}(\lambda)}}}}} \\({vii}) & {{{WFE}(\lambda)} = \frac{{{{s_{i3}(\lambda)} - z}} - \sqrt{\left( {{s_{i3}(\lambda)} - z} \right)^{2} - y^{2}}}{\lambda_{ref}}} \\\quad & {\quad {= {\frac{1}{\lambda_{ref}}\left\lbrack {{{f_{3} - \frac{f_{3}^{2}}{\Delta \quad {s_{i2}(\lambda)}} - z}} - \sqrt{\left( {f_{3} - \frac{f_{3}^{2}}{\Delta \quad {s_{i2}(\lambda)}} - z} \right)^{2} - y^{2}}} \right\rbrack}}}\end{matrix}$

[0086] e) Number Run:

[0087] (i) s_(oi)=100 mm

[0088] (ii) λ_(ref)=1.4 μm

[0089] (iii) d=260 mm

[0090] (iv) y=10 mm

[0091] (v) z=50 mm

[0092] (vi) ƒ₁=ƒ₂=η₃=50 mm

[0093] (vii) N₀(λ_(ref))=1.3

[0094] (viii) N_(ave)(λ)=1.302

[0095] (ix) Δs_(i2)(N_(ave)(λ))=0.03691172 mm; ∴

[0096] WFE(λ)≈0.53 Waves@ λ_(ref)

[0097] Most wavefront sensors can easily measure errors to less than 1wave, and given a small change of index, there is typically asignificant change in the wavefront error produced. For the above case,where there is a 0.002 index change, the WFE is easily measurable.

[0098] Other properties that can be calculated include any physicalproperty that has a relationship that changes with the refractive index.The refractive index relates to the interaction of light with theelectrons in a substance, the more electrons, and the more polarizablethe electrons, the higher the refractive index. Although viscosity isresistant to the shearing force, it is related to the interactionsbetween molecules as they move past one another. It is possible torelate viscosity and other properties to the refractive index of lightwithin a specific class of components, specifically proteins forexample, by correlating the two properties and using the relationship.For example, for proteins, the viscosity increases because there is moreopportunity for them to interact as they are moving past each other, andthe refractive index also increases slightly because the density ofelectrons is a little higher. For this very restricted class, acorrelation can be made that is valid for that class of proteins.Similar correlations could be made for other non-immiscible components.

[0099] In order for these equations to be solved, it is necessary thatthe fluid components do not chemically interact, such that thebiological component may be separated with the component ratiospreserved. For example, when pure water is a reference fluid, the focalpoint changes as a function of the material in the flow stream. Thelight beam will curve (spread) when compared to the reference fluid.This curvature can be measured. There are a number of combinations thatcan be solved including a refractive index or a ratio of non-immisciblebiological component fractions. If there are two or more unknowns thenadditional wavelengths will be required to solve for the unknown.

[0100] The Shack-Hartmann Wavefront Analyzer is constructed by placingan array of apertures in front of a charge-coupled device or CCD camera.These apertures allow light be diffracted by the plate onto the CCD. Thesegments of the beam that pass through the apertures will be spatiallydisplaced from the center position, based on the direction of travel, orthe phase of that part of the beam. The CCD camera measures the phase ofeach spot by measuring this displacement. Software algorithms thenreconstruct a wavefront for the entire beam. The spacing of theapertures defines the resolution of the system, and the size of eachaperture is calculated to optimize sensitivity to phase changes. Incontrast, a Shack-Hartmann Wavefront Analyzer uses an array of smalllenslets to collect all of the beam in each aperture position, andproject all of it onto a detector.

[0101] Essentially, a spherical wavefront is refracted through thebiological mixture 12, which will eventually be focused. It is preferredthat the focus be located within the biological mixture 12. A keycomponent is the aperture or spatial fluid 26 which eliminates themajority of the (waste) scattered light outside of the focus region. Theaperture or spatial filter 26 functions as a noise filter. This is howconfocal microscopy works. Additionally, the aperture size is optimizedto account for focus shifts (+or −) due to average volume index changes.Any wavefront can be propagated through the test region, if thewavefront is pre-determined before being transmitted through thedistortion zone (e.g., a component-water mix), and if there is areference volume of material (e.g., water) to make a comparison with. Adistortion dependent shift in focus (defocus) is going to be the largestdistortion component, hence, the easiest to detect and measure (even ina noisy environment).

[0102] Not only can the refractive index or relative fractions ofcomponents be calculated but other relative functions like thickness,size, geometry, and viscosity of the cellular components such asdifferent fluids or gels such as the protein matrix-based gels.

[0103] Concerning a flow rate measurement method, a strobe will be usedas shown in FIG. 7 and accommodations made for the boundary effects inthe container or flow tube. The flow profile can be compensated bytaking the flow rate at the center of the container or flow tube and atthe edges and averaging, or testing at the center. LED's are strobed atdifferent duty cycles until particles appear stationary (within acertain tolerance). Hence, the velocity of the fluid can be determined.The sensing array can have a central imaging lens to detect the flowrate and wavefront sensor lenslets to detect the wave front informationand distortions. With a fixed imaging optic, the device measures thevelocity of particulate matter in the focus region in the fluid using astrobe. If the fluid ratios and component values are known, the volumefluid flow rate can be calculated if the center flow rate has beendetermined. By varying the gate time of the strobe, imaged particles mayappear stationary once the gate time is correct.

[0104] With a fixed imaging optic, the device measures velocity ofparticulate matter in the focus region in the fluid using a strobe. Withknowledge of the fluid ratios, and density values, the fluid volume flowcan be determined. It is also possible to scan the imaging optic (usinga speaker coil mounted optic as used in CD players) and collect a rangeof flow data.

[0105] A number of basic improvements result, which include:

[0106] a) reduction of errors due to optical scattering losses;

[0107] b) simplification of instrument calibration;

[0108] c) improved accuracy for low-water-cut (higher ratio ofbiological component to water).

[0109] d) elimination of calibration step;

[0110] e) accurate multi-component detection system over all ratios; and

[0111] f) flow measurements (if required).

[0112] While the invention has been described in connection with apresently preferred embodiment thereof, those skilled in the art willrecognize that many modifications and changes can be made thereinwithout departing from the true spirit and scope of the invention, whichaccordingly is intended to be defined solely by the appended claims.

1. A method of determining the amounts of first and second non-immiscible components in a mixture having known, different indices of refraction in the mixture comprising: a) passing a focused light beam through the mixture; b) measuring the displacement of the actual point of focus from a known focal point through a material with a known index of refraction; and c) calculating the amounts of the first and second components present from the displacement.
 2. The method of determining the amounts of the non-immiscible components present in a mixture as set forth in claim 1, comprising: a) passing a focused light beam having at least two discrete wavelengths through the mixture; and b) separately measuring the displacement of the actual point of focus from a reference focal point for each of said at least two discrete wavelengths.
 3. The method of determining the amounts of biological components present as set forth in claim 1, comprising: filtering the emergent light beam to remove higher order distortions.
 4. The method of determining the amounts of biological components present as set forth in claim 1, comprising: collimating the beam after it passes through the biological components and before measuring the displacement of the focal point.
 5. The method of determining the amounts of biological components as set forth in claim 4, in which measuring the displacement comprises measuring the shape of a collimated wavefront beyond the point of focus.
 6. A method for measuring characteristics of a biological system having at least two contrasting density components comprising: a) projecting light of wavelength λ₁ through a multi-density system to distort wavefront of wavelength λ₁; b) projecting light of wavelength λ₂ through a multi-density system to distort a wavefront of wavelength λ₂; and c) determining biological components information in response to the distortion of the light of wavelengths λ₁ and λ₂.
 7. The claim of claim 6, wherein the biological information determines the refractive index of a biological component.
 8. The claims of claim 6, wherein the biological information determines the ratio of biological components.
 9. The claim of claim 6, wherein the biological information determines both the refractive index and the ratio of biological components.
 10. A method of analyzing biological components in a system, having a plurality of contrasting density components comprising: a) a projecting light comprising λ₁ and λ₂ through the fluid stream such that (i) λ₁: chosen such that N_(BC) (λ₁)≠N_(water) (λ₁) and N_(BC) (λ₁)≠N_(air)(λ₁); (ii) λ₂: chosen such that N_(water) (λ₂)≠N_(water)(λ₂) and N_(BC)(λ₂)≠N_(air)(λ₂); b) projecting light through the system such that the focus indicates a specific biological component; c) further projecting light through an aperture and collimating lens array to an area sensor; and d) detecting the wavefront distortions to determine the specific biological components.
 11. The claim of claim 10, further comprising using a strobe to determine movement of the components.
 12. The claim of claim 10, further comprising tuning the detector to defect a plane wave when projected through water.
 13. A biological imager for measuring the biological components in a system comprising: a) a light source comprising wavelengths λ₁ and λ₂ such that when projected through the biological sample, produces a distinct signature due to the distortion of the wavefront; and b) detection to sense the signature.
 14. The claim of claim 13, further comprising using a Shack-Hartmann Wavefront Analyzer.
 15. The claim of claim 13, further comprising solving the first order differential equations.
 16. The claim of claim 13, such that a focus moves left or right to indicate more or less of a component. 